Jan van der Ploeg

Genetics and biochemistry of 1,2-dichloroethane degradation

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the α/β hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.

Transformation of Isopropylamine to l-Alaninol by Pseudomonas sp. Strain KIE171 Involves N-Glutamylated Intermediates

ABSTRACT Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or l-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with d-alaninol. To investigate the hypothesis that l-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and l-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to l-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of γ-glutamyl-isopropylamide from isopropylamine, ATP, and l-glutamate, was shown to be catalyzed by IpuC, a γ-glutamylamide synthetase. γ-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding γ-glutamyl-l-alaninol [γ(l-glutamyl)-l-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate l-alaninol and to regenerate l-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, l-alaninol is oxidized by an alcohol dehydrogenase to yield l-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either l-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

Sequence analysis of the upstream region of dhlB, the gene encoding haloalkanoic acid dehalogenase of Xanthobacter autotrophicus GJ10

The DNA sequence upstream of thedhlB gene encoding the haloalkanoic acid dehalogenase ofXanthobacter autotrophicus GJ10 was determined and contained an open reading frame, designateddhlC, which encoded a protein with a significant similarity with the family of Na+-dependent symport proteins. ThedhlC gene was subcloned under control of a T7 promoter, and found to encode a polypeptide of 45 kDa on SDS-PAGE. Upstream ofdhlC, a −24/−12 promoter sequence was found. Further upstream, in the opposite direction of transcription, another open reading frame, designateddhlR, with homology with the family of σ54-dependent transcriptional activator proteins was detected. ThedhlR gene was cloned and expressed under the control of a T7 promoter and encoded a polypeptide of 51 kDa on SDS-PAGE. The genetic organization of thedhlB region suggested that the expression ofdhlC anddhlB was controlled by the product ofdhlR and σ54 which may explain the observed overexpression of the haloalkanoic acid dehalogenase under starvation conditions.

Adaptation of Pseudomonas sp GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while Vmax values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde.